Design | BNUZH-China

Design

Overview

Chassis strains

Strategy 1 : Degradation of PE plastics by Pseudomonas aeruginosa PAO1

Module 1 : Binding and Degradation

Module 2 : Assimilation and Mineralization

Module 3 : Deep degradation of Pseudomonas aeruginosa PAO1

Strategy 2 : Extracellular Electron and CO₂ Transfer

Module 1 : Biological Synthesis of High-Conductive Pili in Pseudomonas aeruginosa PAO1

Module 2 : Electron Transport Optimization - nqrf Gene Upregulation

Module 3 : Conversion between CO₂ and Bicarbonate

Strategy 3 : Carbon Fixation of Rhodopseudomonas palustris CGA009

Module 1 : De novo biosynthesis of NAD⁺

Module 2 : Biosynthesis of NADP⁺

Module 3 : Promotion of NADPH conversion

Module 4 : Synthesis of cellulose

Suicide System

References

Overview

Microplastics, emerging as a new type of pollutant, are ubiquitous in marine environments and pose a severe threat to ecosystems, particularly to mangroves. These crucial ecological barriers are at risk due to the accumulation of microplastics, which can harm the health of mangroves and impact local ecology and economy. To address this issue, we are employing synthetic biology to enhance the ability of Pseudomonas aeruginosa PAO1 to degrade microplastics, improving the bacterium's adsorption, degradation, and mineralization processes to tackle the challenge of degrading microplastics in deep soil. Additionally, to prevent the negative impact of carbon dioxide produced during the degradation of PE microplastics on the carbon sequestration capacity of mangroves, we have introduced Rhodopseudomonas palustris CGA009 capable of absorbing carbon dioxide and performing the Calvin-Benson-Bassham (CBB) cycle, thereby fixing excess carbon in the mangrove soil and enhancing the ecological effect of mangroves.

Chassis strains

We chose Pseudomonas aeruginosa PAO1 as the chassis. P. aeruginosa is a common bacterium in mangrove ecosystems, so compared to foreign species, the use of native species does not cause excess gene leakage and does not have a significant impact on the ecosystem, ensuring its effectiveness. P. aeruginosa is able to utilise a wide range of carbon substrates as a source of carbon and energy. It thrives over a wide range of temperatures, is ubiquitous and has the potential to form biofilms in the environment, thereby enhancing the degradation of polyethylene microplastics.

Rhodopseudomonas palustris CGA009, a widely studied and used biological chassis, is a bacterium with the properties of extraordinary metabolic versatility, carbon source diversity and metabolite diversity. It indicates that R. palustris CGA009 can use CO₂ as the carbon source, which is fixed by the Calvin Bassham Benson (CBB) cycle to participate in cell growth metabolism. Furthermore, many researches indicates the potential of R. palustris as a chassis organism for biopolymers and their building blocks production, such as polysaccharide. In addition, after an electrosyntrophic coculture is formed, R. palustris CGA009 can grow autotrophically under dark, anoxic conditions through syntrophic interspecies electron transfer.

Strategy 1 : Degradation of PE plastics by Pseudomonas aeruginosa PAO1

Biodegradation is mainly divided into 4 steps:
(1) Bio-deterioration: microbial communities and abiotic factors work together to cut polymers into fragments;
(2) Depolymerization: microorganisms secrete enzymes and free radicals to convert polymers into oligomers, dimers or monomers;
(3) Assimilation: the depolymerized molecules are recognized by the receptors on the surface of the microorganism and cross the plasma membrane into the microbial cell;
(4) Mineralization: The depolymerized molecules are metabolized and oxidized into small molecule compounds such as CO₂、N₂、CH₄ and H₂O in the microbial cells. In our project the PE degradation ability of P. aeruginosa will be improved according to the above four steps.

Module 1 : Binding and Degradation

We engineered two parts of the passenger protein: the PE-binding peptide and the PE enzyme. We used modelling to predict the sequence of the PE-binding peptide, whose hydrophobic and van der Waals forces help our engineered bacteria bind to the microplastic surface and enhance the ability of P. aeruginosa to aggregate near the microplastic. The most limited step in the plastic degradation process is actually the degradation of microplastics from polymers to monomers, so we fused an enzyme capable of depolymerising PE downstream of PEBP. Tianjin University has verified that this enzyme can be expressed in Escherichia coli and play a good role in extracellular environment last year, so we chose this enzyme as our PE degrading enzyme.We chose PEase (from Tianjin University), an enzyme found in the saliva of waxworms that oxidises PE into PE monomer.

Figure 1 Passenger Protein Schematic

To immobilise these two proteins on the bacterial surface, we chose autotransporters as the anchoring method and predicted the signal peptide and β-barrel region of the estA protein of P. aeruginosa. As part of the transporter, they bind to both ends of our passenger protein so that our protein is well anchored to the bacterial outer membrane. In addition, we added a linker, tags, the enzyme cutting sites and other components in the middle of these modules to facilitate the expression, purification and transformation of our components.

Figure 2 Two proteins are immobilized on the bacterial surface

Module 2 : Assimilation and Mineralization

We construct AlkB2-Rd45-Adh fusion protein. Membrane-bounding Alkane hydroxylase (AlkB2) can hydroxylate straight alkanes to alcohols and transport it into the cytoplasm, which improves the ability of P. aeruginosa to assimilate alkanes and degrade PE plastics. As AlkB2 needs one or more redox partners to obtain electrons for monooxygenation reactions, we link rubredoxin Rd45 from alkane hydroxylase-rubredoxin fusion gene alkW1 in Dietzia sp. DQ12-45-1b after AlkB2. This protein enables AlkB2 to play a better role without coupling more rubredoxin proteins.

Figure 3 Fusion Protein Effect Diagram
(A) Degradation Pathway of AlkB2-Rd45-Adh Fusion Protein (B) Degradation Pathway of CYP-VHB Fusion Protein

The pathway that now has been proposed for the degradation of n-alkane includes initial hydroxylation at the terminal ω carbon by a hydroxylase like AlkB2 or CYP, further followed by the formation of the corresponding ketone by a primary alcohol dehydrogenase (Adh). Besides, studies have shown that AlkB will consume NADH while Adh will consume NAD⁺ and produce NADH in catalyzation. In order to facilitate the further degradation of n-alkane and reproduction of NADH, we fuse AdhA after the Rd45 protein.

AlkB along with Rd45 and Adh construct a rather complete assimilation and mineralization module. However, AlkB is known for hydroxylate terminal or subterminal carbon on long-chain and medium-chain alkane but not short-chain alkane. To make up for the insufficient hydroxylation ability of AlkB to short-chain alkane and the inability to conduct in-chain hydroxylase. Therefore, we also introduced P450camY96F enzyme that can degrade short-chain alkane and conduct in-chain hydroxylate into P. aeruginosa. It has been proved that this enzyme can greatly accelerate the degradation efficiency of PE in the case of heterologous expression.

Figure 4 Assimilation and Mineralization module

Module 3 : Deep degradation of P. aeruginosa

Studies have shown that microplastics in mangrove soil sediments are vertically distributed at a depth of 0-100cm, and our chassis are aerobic symbiotic bacteria attached to the roots of mangrove plants, and oxygen is required for the degradation of PE, so the degradation of microplastics in deep soil and anaerobic areas that may be caused by tides faces problems.

VHb is a single-domain hemoglobin (SDHb) which can efficiently bind oxygen and transport it to the respiratory chain by interacting with terminal oxidases, especially under oxygen-restricted conditions. In this way, VHb significantly enhances the regeneration of NAD⁺, which is one of the substrates of most of our monooxygenase. In addition, VHb can also interact with transcriptional regulators responsible for oxygen responses, triggering oxidative phosphorylation in cell.

Figure 5 Deep degradation of P. aeruginosa

In this regard, the transfer of the vgb gene of the bacterial oxygen-carrying protein VHb enabled: enhancement of the enzymatic activity of the polyethylene mineralisation pathway of the engineering bacteria to accelerate microplastic degradation and endowment of the engineering bacteria with anaerobic environmental resilience to ensure that they can degrade plastics in anaerobic zones deep in the soil. Ultimately, full coverage of the breadth and depth of plastic degradation by engineering bacteria in mangroves will be achieved.

Strategy 2 : Extracellular Electron and CO₂ Transfer

P. aeruginosa PAO1, a thriving microbe in bioelectrochemical systems (BES), produces versatile phenazine redox mediators. BES, especially microbial fuel cells (MFCs), are rapidly developing for renewable energy and bioremediation. MFCs generate electricity via extracellular electron transfer from microbes degrading organic matter.

The most commonly described transfer mechanisms are direct electron transfer via direct cell contact or protein nanowires and mediated electron transfer via secondary or primary metabolites.

Attempts to improve the biological efficiency of MFCs have therefore focused on understanding and improving these mechanisms. In mediated electron transfer, microorganisms utilize endogenous or exogenous soluble redox mediators that enable transmission of electrons to an external electron acceptor. In bacteria, endogenous secondary metabolites used as mediators include riboflavins in Shewanella, henazines in P. aeruginosa, and quinones in Lactococcus. These molecules undergo reversible oxidation and reduction and hence can be used repeatedly as electron shuttles.

In mixed microbial communities and biofilms, redox mediators may be shared among different species, potentially fostering syntrophic interactions. Naturally, microbial consortia are formed within these communities, characterized by complex interactions that often enhance resource utilization. Further research has revealed synergistic relationships involving both native redox mediators and non-redox mediator producers, with these effects being especially significant under oxygen-limited conditions. P. aeruginosa-derived redox mediators have been demonstrated to facilitate extracellular electron transfer in a synergistic interaction with other strains. This provides a good basis for the linkage design with R. palustris in the deep soil in our project.

So one of the key challenges in enhancing the performance of extracellular electron transfer (EET), particularly for the strain P. aeruginosa PAO1, is improving the efficiency of microbial electron transfer to the anode and the number of microbial nanowires.

We degraded PE microplastics in mangrove soils using engineered P. aeruginosa .
However, microplastics and their biodegradation release CO₂, altering water nutrients, decreasing microbes, and impacting carbon-nitrogen cycles.

We employed R. palustris CGA009, abundant in mangroves, as an auxiliary to absorb CO₂ from PE degradation by P. aeruginosa. Leveraging its CO₂ uptake, we modified R. palustris to optimize carbon absorption, minimizing emissions during degradation.

Module 1 : Biological Synthesis of High-Conductive Pili in P. aeruginosa

We will construct a truncated version of the PaPilA gene containing only the first 61 amino acids of the N-terminus, and perform a PaPilA1–61M₃ plasmid with three amino acid mutations (E32Y, L51F, and G57Y) to greatly increase the biopotential output of P. aeruginosa.

Module 2 : Electron Transport Optimization - nqrf Gene Upregulation

Researches show that phenazine reductase activity in membrane fractions is attributable to the three NADH dehydrogenases present in P. aeruginosa and that their order of phenazine reductase activity is Nqr > Nuo > Ndh. Besides, the Nqr complex plays a role in the extracellular electron transport (EET) process of P. aeruginosa, particularly under anaerobic conditions, by promoting the reduction of phenazines to facilitate energy production and the establishment of proton motive force.

Therefore, by increasing the activity of Nqr, the electron transport capacity of P. aeruginosa can be improved.

Figure 6 Reduction of phenazine compounds promotes energy production

Module 3 : Conversion between CO₂ and Bicarbonate

CO₂ is slightly soluble in water at room temperature (about 600:1) and produces mainly carbonic acid in water, but also small amounts of carbonate and bicarbonate. Therefore, carbon dioxide will be released from the water body under heating and shocking, so the carbon dioxide released by engineered bacteria in mangrove environments will escape from the water volume easily; in addition, during the process of degradation of P. aeruginosa and R. palustris will form biofilms on the surface of microplastics, which CO₂ transfer will be severely hindered. Therefore, we need to transform CO₂ into a kind of substance that can be easily delivered in biofilms.

Dissolved inorganic carbon (DIC) toolkit is a system composed by inorganic transporter and carbonic anhydrase (CA) in autotrophs using Calvin Benson Basel cycle (CBB cycle). We create the DIC toolkit in R. palustris CGA009 and the delivery relationship between the two engineered bacteria.

Figure 7 The schema of the DIC toolkit in the engineered bacteria and interactions

P. aeruginosa PAO1 can encode three cytoplasmic β-CAs: psCA1, psCA2, and psCA3. CAs can catalyze the reversible conversion of CO₂ to bicarbonate: $$CO_2 + H_2O ⇌ HCO_3^- + H^+$$

Among them, psCA1 (encoded by the gene PAO102 or psCA1) is a β-carbonic anhydrase of type I, which has the highest conversion efficiency among the three and its secondary structure is not affected by pH.

It's known that there is the Bicarbonate transporter BicA (from Bicarbonate transporter BicA [Pseudomonas aeruginosa] - Protein - NCBI (nih.gov) ) on the membrane of P. aeruginosa PAO1. We take advantage of psCA1 to transform CO₂ that is produced by P. aeruginosa PAO1 during the process of PE degradation to HCO₃^-, and later transport HCO₃^- out of the cell via BicA.

R. palustris CGA009 is a type of gram-negative bacteria that contain two cellular compartments (cytoplasm and periplasm). An α-CAs (encoded by acaP) is present in its periplasm. It has been shown that this kind of CA is inactive in aerobic environment and active in anoxic environment. Microplastics in mangroves are distributed in low-oxygen environments such as water and soil. In R. palustris CGA009, the function of this kind of CA is to accelerate the speed of conversion from HCO₃^- to CO₂ when the inorganic carbon source is bicarbonate, and fix CO₂ by ribulose-bisphosphate carboxylase-oxygenase (RubisCo), finally conversing to utilizable organic matter.

There is bicarbonate membrane transporter named ABC transporter substrate-binding protein existing in the cell membrane of R.palustris CGA009 (from ABC transporter substrate-binding protein [R.] ). After HCO₃^- taken in engineered bacteria, α-CAs catalyzes the process of HCO₃^-/ CO₂ conversion to produce CO₂ for CBB cycle after caught by RubisCo.

In order to process as much CO₂ as possible from the decomposition of microplastics, we need to increase the efficiency of engineered bacteria in HCO₃^-/CO₂ conversion. Given that the structures of all three CAs in P. aeruginosa PAO1 are highly conserved and adjusting the structure can easily lead to inactivation. Accordingly, we apply the method of psCA1 multiple copies to produce bicarbonate more efficiently.

For the same reason, in order to enhance the efficiency of the conversion from HCO₃^- to CO₂, we still take the approach of multiple copies of acaP.

In addition to some parts of bicarbonate delivered to R. palustris CGA009, some of them also combines with Ca²⁺ inside and outside the engineered P.aeruginosa PAO1 to form CaCO₃ deposits.
However, it has been shown that:
(1) Calcium carbonate deposition is easy to form when the water body is in calm and a high concentration of CO₂ exists in it, but little calcium deposition occurs when the water is in waving and in a common CO₂ conditions;
(2) If calcium carbonate deposition is produced, it also can contribute to the stabilization organic matter and reduces the amount of carbon dioxide released from soil.
(3) And even the small amount of calcium carbonate is produced, it is also important in the formation of biofilm. Therefore, the formation of calcium carbonate deposits is not considered as an issue in our project.

Strategy 3 : Carbon fixation of Rhodopseudomonas palustris CGA009

R. palustris is renowned for its ability to function in the four known metabolisms of life. This unique ability allows R. palustris to convert light or electrical energy into chemical energy in organic matter. It suggests that R. palustris can drive the fixation of carbon dioxide and the synthesis of bacterial cellulose through syntrophic interspecies electron transfer.

As a precursor of bacterial cellulose, the synthesis of glucose requires glyceraldehyde-3-phosphate. However, the intracellular level of NADPH limits the efficiency of the Calvin cycle of R. palustris in producing glyceraldehyde-3-phosphate. Since the Calvin cycle is constrained by the concentration of NADPH and NADP⁺, the synthetic biological strategy to improve the intracellular NADPH and NADP⁺ level is of great value to prompt the efficiency of Calvin cycle.

Figure 8 The schematic of the carbon fixation

Module 1 : De novo biosynthesis of NAD⁺

The lack of NAD⁺ may have an adverse effect on R. palustris CGA009. Nicotinamide mononucleotide (NMN) is an important precursor of NAD⁺, and R. palustris CGA009 cannot directly synthesize NAD⁺ from NMN, and the synthesis efficiency of NAD⁺ is low. Therefore, we introduced the gene encoding nicotinamide-nucleotide adenylyltransferase NadM of nadK from Francisella tularensis SCHU S4 to convert NMN directly to NAD⁺ and avoid depletion of NAD⁺.

Module 2 : Biosynthesis of NADP⁺

Because phosphorylation of NAD by NAD kinase (NADK) is the only known mechanism by which NADP is produced de novo3, in order to increase NADP⁺ synthesis, we introduced the gene encoding NADK of nadK from Rhodopseudomonas palustris RCB100 to increase NADP⁺ synthesis.

Module 3 : Promotion of NADPH conversion

In addition to the above two modules, we introduced the gene encoding PntAB of pntA and pntB from E. coli MG1655 to increase the original rate of the conversion of NADP⁺ to NADPH. Ultimately, since the biosynthesis of glyceraldehyde-3-phosphate depends on the intracellular level of NADPH, module 1-3 will promote the production of glucose.

Figure 9 The introduction of NADPH biosynthesis pathways in engineered bacteria

Module 4 : Synthesis of cellulose from R. palustris

In the microbial carbon sequestration pathway, carbon is usually accumulated through refractory substances in biological residues, so increasing the synthesis and accumulation of cellulose of refractory substances can effectively retain carbon in the soil after bacterial death.

Cellulose synthesis usually involves multiple subunits, of which BcsA and BcsB are the core catalytic subunits responsible for the synthesis of cellulose chains. Therefore, we increased cellulose synthesis by transferring to bcsa and bcsb derived from Pseudomonas pactida NBRC 14164.

Suicide System

The overview of the system of hok/sok

Suicide after the completion of the task by the engineered bacteria is an important module to ensure the safety of the project.

1. The mechanism of the hok/sok system

The hok/sok system is a type I TA. Antisense RNA, which acts as an antitoxin, binds to the mRNA encoding toxin proteins, inhibiting translation of the toxin proteins and promoting degradation of the toxin-antitoxin complex.
The locus consists of three genes:
(1) the host killing gene (hok) encodes a highly toxic transmembrane protein, which can lead to the collapse of cell membrane potential, cellular respiratory arrest, cell shape changes, and ultimately cell death;
(2) the modulation of killing gene (mok) is overlapped with hok and encodes the lead peptide of the hok protein;
(3) the suppression of killing gene (sok) is able to express a cis-encoded small antisense RNA that binds to the mok RNA and causes the complex to be enzymatically degraded, leaving mok incapable of expressing the hok lead peptide and unable to produce hok toxin proteins. The sok is driven by a strong promoter but hok/mok is driven by a weaker promoter so that there will be more sok antisense RNA than mok RNA in the normal cell and the toxin cannot be expressed; however, the half-life of sok antisense RNA (~30s) will be much shorter (20-30min) than mok RNA. The schema of the mechanism is shown below.

Figure 10 The schema of hok/sok system

2. The application of plasmid-loss prevention

The application of hok/sok system in preventing plasmid loss is a kind of post-segregational killing (PSK). We still face the problem of plasmid loss after the plasmid is transferred into the engineered bacteria due to plasmid instable replication, burden effect, mutation and recombination, etc. Therefore, we introduce the hok/sok system to prevent plasmid loss.

In our project we introduce the toxin-antitoxin system (TA) from the plasmid R1 of Escherichia coli. Assuming that the recombining plasmid in engineered bacteria is accidentally lost, both hok and sok can no longer express in the bacteria. However, since the degradation rate of sok antisense RNA is much faster than that of mok RNA, the remaining pool of mok RNA in the cell can express after all the sok antisense RNAs are all degraded. Then hok toxin proteins can be produced to cause the death of the engineered bacteria that have lost the plasmid. That is able to keep the plasmid stable in the engineered bacteria.

3. The application of suicide systems after departure from environments with high concentration of citrate

Influenced by the root exudate of mangrove, the mangrove peripheral environment exists a high concentration of citrate. Therefore, we can choose the citrate concentration perceived by the engineered bacteria as the basis for judging whether the engineered bacteria leave the mangrove environment or not.

Here we replace the promoter of the hok/mok locus with the common weakly constitutive promoter PcW from P. aeruginosa. Moreover, we replace the promoter of sok with the promoter P_opdH from opdH-tctCBA-tctDE manipulator system. When there is not citrate in the peripheral environment, the tctD from opdH-tctCBA-tctDE manipulator system will specifically binding to P_opdH and inhibit its activity; however, when the citrate is present, tctD will bind to citrate and no longer repress the promoter. Besides, the tctE from opdH-tctCBA-tctDE manipulator system can also ease the inhibitory effect of tctD through extracellular sensing of tricarboxylates. The effect of the two aspects leads to the restoration of P_opdH activity and downstream gene expression.

As mentioned above, when citrate is present in the environment (the engineered bacteria haven't left the mangrove area), sok can be expressed and produce sok antisense RNA that bind to the mok RNA, so that the toxin proteins cannot be expressed; once the engineered bacteria leave the mangrove area, the citrate concentration decreases, which leads to the decrease in the activity of the P_opdH promoter. The expression of sok decreases or even stops, and the sok antisense RNA is rapidly degraded, which leads to the release of mok RNA. The hok toxin protein is able to be expressed, resulting toxicity to the cells (the principle is similar to that of the plasmid loss). Therefore, after the engineered bacteria accidentally leaves the mangrove environment, it is able to initiate suicide thus avoiding biological invasion, genetic contamination and other situations.

A schematic diagram of the above two points is shown below.

Figure 11,12 The schema of the hok/sok system for non-citric suicide and plasmid-loss prevention

4. Reasons for choosing the system from E.coli R1 plasmid

(i) The hok/sok locus present on other plasmids of E. coli are inactivated probably due to insertion sequences, point mutations or larger rearrangements.

(ii) A homologous structure of the hok/sok locus exists in P. aeruginosa PAO1, but its function in intoxicating cells remains unproven.To ensure the experimental rigour, we do not use this locus.

(iii) E. coli and P. aeruginosa are both in the γ-proteobacteria order, which makes them own more similar physiological characteristics and leads to good systemic effect.

5. Reasons for choosing P_opdH from opdH-tctCBA-tctDE manipulator system

(i) This system was originally present in P. aeruginosa and R. palustris, which is no need to introduce additional regulatory elements into the engineered bacteria.

(ii) P. aeruginosa use tricarboxylic acids (e.g., citric acid) as the main carbon source. The catabolite repression control (Crc) system can also be used as a regulatory system in our design; however, it will cause an increased preference for the main carbon source like citric acid and inhibit the use of secondary carbon sources, which is unfavorable to the specific control when the engineered bacteria are in the environments with high citric acid concentration.

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